This laboratory has identified Chloride Intracellular Channel 4 (CLIC4) as an important mediator of cell cycle arrest and apoptosis. CLIC4 expression is regulated by p53, c-Myc and TNF alpha, proteins that control cell cycling in numerous tissues and tumors. An important aspect of CLIC4 biology in keratinocytes and other cell types is the common translocation from the cytoplasmic to the nuclear compartment under various conditions of cell stress that lead to cell cycle arrest or apoptosis. Since CLIC4 has a functional nuclear localization signal within its sequence, nuclear translocation is considered a physiological event. We discovered that CLIC4 expression is upregulated in differentiating mouse and human keratinocytes where it translocates to the nucleus early in the maturation process. Knockdown of CLIC4 prevents expression of keratinocyte differentiation markers and abates the cell cycle arrest associated with maturation. Conversely, introducing nuclear CLIC4 into basal keratinocytes via a targeting vector induces growth arrest and expression of differentiation markers. Thus CLIC4 mediates and is required for keratinocyte differentiation. Nuclear translocation of CLIC4 is impaired in many epithelial cancer cell lines and tumors, suggesting that altering CLIC4 biology might be important for the pathogenesis of malignant disease. A yeast two-hybrid screen with CLIC4 as bait was performed and identified Schnurri-2 as a binding partner. Schnurri-2 is known to mediate TGFbeta dependent transcription in Drosophila and mammals. Schnurri-2 interacts within aa 121-197 of CLIC4 and is required for CLIC4 nuclear translocation. TGFbeta treatment enhances the physical association of CLIC4 and Schnurri-2 and increases both CLIC4 and Schnurri-2 protein expression and nuclear translocation. In turn, nuclear CLIC4 enhances TGFbeta signaling when examined by reporter assays, cell cycle changes and protein expression. These studies are the first to assign a mechanism for CLIC4 mediated cell cycle or cell viability effects, and identify nuclear CLIC4 as an important component of the TGFbeta pathway. A mechanism of action through TGFbeta may explain both the influence of CLIC4 on keratinocyte differentiation and the altered biology of CLIC4 in tumor cells where TGFbeta signaling is known to be impaired. While coupling to Schnurri-2 enhances CLIC4 nuclear transport, the molecular basis by which multiple stimuli encourage nuclear localization has been an enigma. Clarification comes from studies showing that diverse nitric oxide donors induce CLIC4 nuclear translocation. Further, TNFalpha-induced CLIC4 nuclear translocation is concurrent with an increase in NO levels, and pretreatment with NOS2-specific inhibitor 1400W inhibits TNFalpha-induced CLIC4 nuclear translocation. Nuclear translocation of CLIC4 is independent of cGMP-dependent NO signaling. Rather, increased nuclear translocation of CLIC4 in diverse cell types is coincident with increased S-nitrosylation of the protein on specific cysteine residue(s) and is significantly decreased by pretreatment with 1400W. Cysteine &#61614; alanine point mutation at residue 35 decreases protein stability by proteasome-dependent degradation while Cys &#61614; Ala mutation at residue 234 shows increased cytoplasmic residence. These studies indicate that redox potential changes and generation of NO provide a common environment that regulates CLIC4 subcellular localization in response to multiple extrinsic stimuli. By virtue of it extraordinary size and external location, the skin serves as a first line immune organ, and keratinocytes express a large number of cytokines and chemokines. In cases where control of these factors is abnormal, they serve as intermediaries in multiple inflammatory conditions of the skin. Therefore, understanding the normal regulation of cutaneous cytokines and chemokines is important for both protecting the host and for therapeutic approaches to disease. Cutaneous Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) is upregulated in atopic dermatitis and psoriasis, two common and disabling inflammatory skin diseases. In human and mouse keratinocytes, cytokine induced expression of GM-CSF is dependent upon epidermal growth factor receptor (EGFR) activity. Furthermore, ablating the EGFR genetically or pharmacologically inhibiting the EGFR or its downstream effectors in the MAP kinase pathway prevents induction of cutaneous GM-CSF. Transcription of the GM-CSF gene is regulated by AP-1 factors, and ablating the EGFR reduces c-jun phosphorylation substantially after cytokine stimulation. These studies provide a novel target through the EGFR and its downstream effectors to treat selected cutaneous inflammatory diseases. We have been exploring another family of potentially proinflammatory factors in the skin. S100A7 and closely related S100A15 are upregulated in psoriasis and other inflammatory cutaneous diseases. S100 proteins are small, calcium-binding proteins constituting the largest, multigenic family of calcium-binding EF-hand proteins. They show distinct tissue and cell type specific expression pattern indicating local specification. The human S100A7 (psoriasin) and S100A15 reside in a chromosomal cluster of highly similar paralogs within the Epidermal Differentiation Complex (Chromosome 1q21; 3). To exploit the power of mouse models for determining functions of gene products, the corresponding S100A7/A15 ortholog was cloned and examined in normal murine skin. The single mouse S100A15 gene encodes a protein of 104 amino acids with a predicted molecular weight of 12,870 Da and two EF-hand calcium binding sites. Murine S100A15 is regulated by protein kinase C alpha, calcium and AP-1 factors and is upregulated in differentiating keratinocytes and incorporated into the cornified envelope. Currently, a transgenic mouse has been generated with inducible S100A15 targeted to the epidermis as a model for S100A7/15 associated cutaneous diseases. hS100A7and hS100A15 are highly homologous and co-expressed in the same inflammatory diseases, but the biological impact of the co-expression of these closely related genes remains undefined. By generating specific antibodies, we demonstrated that hS100A7 and hS100A15 are differentially expressed in specific cell types of human skin. While sequence identity of the proteins is high, crystal structural data reveal distinct spatial patterns in the functionally important homodimer complexes of hS100A7 and hS100A15. Both proteins act as chemoattractants, but their chemotactic activities towards specific leukocyte subtypes are different and are mediated by distinct classes of receptors. hS100A7, but not hS100A15, binds and directly mediates chemotaxis through RAGE (receptor of advanced glycated end products) in both in vitro chemotaxis assays and in vivo in mouse models. hS100A7-RAGE binding, signaling and chemotaxis are zinc-dependent in vitro, reflecting the zinc-mediated changes in the hS100A7 dimer structure. In contrast, hS100A15 promotes chemotaxis through a yet to be identified pertussis-toxin sensitive Gi protein-coupled receptor. The single mouse protein ancestral to both hS100A7 and hS100A15 binds to RAGE and directly stimulates chemotaxis partially but not exclusively through RAGE. Combined treatment with hS100A7 and hS100A15 potentiates the chemotactic response in vivo. Thus, hS100A7/ hS100A15 gene duplications occurring late in evolution have led to biological diversification by which the independent actions of the homologous proteins working through distinct receptors enhance the proinflammatory benefits of the pair but also potentiate their proinflammatory activities in disease